Airway secretory cell fate conversion via YAP‐mTORC1‐dependent essential amino acid metabolism

Abstract Tissue homeostasis requires lineage fidelity of stem cells. Dysregulation of cell fate specification and differentiation leads to various diseases, yet the cellular and molecular mechanisms governing these processes remain elusive. We demonstrate that YAP/TAZ activation reprograms airway secretory cells, which subsequently lose their cellular identity and acquire squamous alveolar type 1 (AT1) fate in the lung. This cell fate conversion is mediated via distinctive transitional cell states of damage‐associated transient progenitors (DATPs), recently shown to emerge during injury repair in mouse and human lungs. We further describe a YAP/TAZ signaling cascade to be integral for the fate conversion of secretory cells into AT1 fate, by modulating mTORC1/ATF4‐mediated amino acid metabolism in vivo. Importantly, we observed aberrant activation of the YAP/TAZ‐mTORC1‐ATF4 axis in the altered airway epithelium of bronchiolitis obliterans syndrome, including substantial emergence of DATPs and AT1 cells with severe pulmonary fibrosis. Genetic and pharmacologic inhibition of mTORC1 activity suppresses lineage alteration and subepithelial fibrosis driven by YAP/TAZ activation, proposing a potential therapeutic target for human fibrotic lung diseases.


23rd Sep 2021 1st Editorial Decision
Dear Dae-Sik, dear Joo-Hyeon, Thank you again for the submission of your manuscript (EMBOJ-2021-109365) to The EMBO Journal. Please accept my apologies for the unusual delay with the peer-review of your work due to protracted referee input at this time of the year and detailed discussions in the team. Your study has been sent to three reviewers with complementary expertise on hippo developmental signaling (referee #1), lung cell biology (referee #2) and amino acid metabolism (referee #3) and we have received reports from all of them, which I enclose below.
As you will see, the referees acknowledge the potential interest of your results and in a timely context, although they also express major concerns, which need to be addressed before they can be supportive of publication at the EMBO Journal. In more detail, referee #2 states that s/he is not convinced of the physiological relevance of the observed YAP/TAZ-induced AT1-NAPT fate acquisition, and requests additional experimentation to corroborate this claim (ref#2, pts.1,2). Reviewer #3 points to concerns on the mechanistic depth presented for the induced TOR-ATF4-EAA axis and its impact on fate conversion and asks you to provide more details (ref#3, pts.1,4). Referee #1 requests a revised consideration of the discrepancies between dKO tissue versus organoid phenotypes (ref#1, pt.2). Finally, the referees list a number of additional issues related to nomenclature applied and technical controls related to the sample purity and potential confounding factors, that would need to be addressed to achieve the level of robustness needed for The EMBO Journal.
Given the referees' overall positive recommendations and detailed constructive comments, I would like to invite you to submit a revised version of the manuscript, addressing the issues raised. As it is EMBO Journal policy to allow only a single round of revision, acceptance of your manuscript will therefore depend on the completeness of your responses in this revised version.
In light of the extensive experimentation requested by the reviewers, I would appreciate if you could contact me during the next weeks via e.g. a video call to discuss your perspective on the comments and potential plan for revisions.
We generally allow three months as standard revision time. As a matter of policy, competing manuscripts published during this period will not negatively impact on our assessment of the conceptual advance presented by your study. However, we request that you contact the editor as soon as possible upon publication of any related work, to discuss how to proceed. Should you foresee a problem in meeting this three-month deadline, please let us know in advance and we may be able to grant an extension.
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Thank you for the opportunity to consider your work for publication. I look forward to your revision.
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Further information is available in our Guide to Authors: https://www.embopress.org/page/journal/14602075/authorguide Revision to The EMBO Journal should be submitted online within 90 days, unless an extension has been requested and approved by the editor; please click on the link below to submit the revision online before 22nd Dec 2021: https://emboj.msubmit.net/cgi-bin/main.plex This is an interesting manuscript. The phenotype of Lats1/2 dKO is carefully examined in the lung bronchial airway. In the Lats1/2 dKO, the columnar secretory cells lose their normal architecture and function, and are transformed into a flattened cell type that resembles alveolar AT1 cells. This fate conversion appears to involve TORC1 and ATF4. The human syndrome bronchiolitis obliterans appears similar to the Lats1/2 dKO phenotype.
Overall, the quantity and quality of data analysis are highly impressive, and the results will be of great interest to the Hippo signalling field, as well as to pulmonary science.
Minor comments 1. The human BO phenotype characterisation is important. Would the authors consider making this Figure 1 and mentioning it in the abstract, or even title of the manuscript? 2. It is interesting that the dKO organoids form multilayered epithelia, while in vivo the dKO forms a monolayer. Can the authors speculate as to why?
Referee #2: In this manuscript, Jeon et al., used elegant genetic and pharmacological models to modulate Yap, mTOR and amino acid uptake mechanisms and claim that activation of Yap in airway secretory cells converts them to alveolar type-1 (AT1) cells via mTOR/amino acid import mechanisms. Specifically, the authors used genetic models to modulate Yap signaling in airway secretory cell and claim that activation of YAP in SCGB1A1+ airway secretory cells convert them into AT1-like cells in vivo and in ex vivo organotypic cultures. Additionally, the authors state that this plasticity involves a recently identified DAPT state through which secretory cells transition to AT1 cells. The authors also claim that YAP-mediated essential amino acid influx controls secretory cell plasticity via mTOR signaling. Finally, the authors claim that similar mechanisms are at play in human lung diseases including pulmonary fibrosis and bronchiolitis obliterans.
In summary, while the mechanisms described here are of potential importance to our understanding of airway cell plasticity, as I detailed below, the observations made here falls short of physiological relevance. Additionally, the data provided here does not support some of the claims (see below). Addressing the following comments would make this manuscript more meaningful and support the claims in this manuscript.
Major comments: 1. The authors show that activation of YAP/TAZ is sufficient to convert airway secretory cells into AT1 cells. However, plasticity observed here is somewhat "artificial" as the current data does not show whether the same mechanisms are involved in injury repair. Recent studies (Strunz et al., 2020) have indicated that Sox2-lineage labeled cells can generate AT2 and AT1 cells after bleomycin injury. Therefore, this reviewer questions the relevance of this finding to injury repair! 2. The secretory cell derived AT1 cells are located in airways, so, they may not have physiological relevance as these cells need to be connected to the vasculature for gas exchange. So, the question is whether there is micro-capillary associated with these AT1 cells? Additionally, prior studies have shown that airway secretory cells can migrate to alveolar regions after damage (ex: flu infection). So, how about activating Yap in secretory cells once they migrate to alveoli after injury (ex: flu infection) to test whether these cells can generate AT1? I suggest the author test this as this will reveal the physiological relevance of secretory cell derived AT1 cells. 3. The authors used the term DATPs. However, the plasticity observed here is due to genetic inactivation of Lats1/2 (and not due to injury/damage alone). It is more appropriate to use reprogrammed cells or Yap-induced transient state. 4. The authors used tdTomato+ cells from Scgb1a1-creER/R26-tdtomato mice for scRNA-sequencing ( Fig. 2 and EV3). However, there are numerous immune, fibroblast and mesothelial cells. This suggests that there are contaminating cells and that might obscure some of the findings (ex: organoid cultures). The authors need to provide additional data to support cell purity or repeat these experiments with pure cell populations. 5. Day-21 Images in Fig-2G' and Fig. EV2E: It appears that the authors used the same images (red and green channels) in both figures. Although this does not affect the conclusions in this manuscript, this might be flagged as "image duplication". 6. In Fig-3F, there are some tdTomato+ cells in EtOH treated condition (top right corner). Is that due to sporadic activation of cre recombinase or is this mouse line leaky? 7. Generally, AT1 cells are localized to luminal side in most organoid cultures (Ex: Barkauskas et al., JCI 2013). However, in Fig-3D', AT1 markers are localized to basal side. Therefore, it is unclear whether the AT1-like cells that the authors observed in these models truly represent resident AT1 cells? Therefore, a comprehensive analysis of AT1-like cells in in vivo and ex vivo models and a direct comparison with resident AT1 cells is required to check the validity of the conclusions. 8. In Fig. 5 and EV6, the authors claim that AT1-like cells are observed in airways of pulmonary fibrosis and bronchiolitis Obliterans lungs. However, the marker used here (AQP5) is not specific to AT1 cells. AQP5 is known to express in airway epithelial cells. Therefore, additional markers such as AGER and PDPN are necessary to support this claim. Additionally, the cells do not appear to show flat and thin morphology as expected for AT1 cells. So, this reviewer doubts the conclusion that they are AT1 cells. 9. It is unclear what the authors are trying to show from the data in Fig. EV6A-C? There is no indication that neither AT1 nor DATP cells have originated from secretory cells. Again, the authors chose to label cluster #4 as DATPs. However, the original study (Haberman et al., Science Advances, 2020) have termed them basaloid cells. Altogether, the current data does not support whether de novo conversion of secretory cells into AT1 cells occurs in these diseases. 10. The authors claim that they identified KDR as a marker of secretory cells. There are other prior studies identified KDR as a marker and they (ex: Jiang et al., Developmental Cell, 2021) should be cited here.
Minor: -In Fig-1B and D, it would be easier for the authors if the panels clearly show the mouse genotype.
Referee #3: Summary: The manuscript by Jeon et al., titled "Airway secretory cell fate conversion via YAP-mTORC1-dependent essential amino acid metabolism" offers some mechanistic understanding of a differentiation program in airway secretory cells, implicating YAP/TAZ signaling and downstream engagement and requirement of mTORC1-ATF4-mediated essential amino acid uptake. Although overall the findings are interesting, the study is well-conceptualized and thoughtfully designed, with well-controlled experiments, additional experimental work is needed to substantiate the conclusions in regard to the involvement/role of mTORC1-mediated ATF4 upregulation and the consequent increase in the expression of SLC7A5 and essential amino acid uptake. Essentially, authors need to link (1) YAP/TAZ signaling with mTORC1 (at least experimentally implicate previously known link), (2) mTORC1 signaling with ATF4, and (3) essential amino acid uptake with the conversion of the airway secretory cells into AT1 cells.
Specific Comments: 1) It remains to be determined how essential amino acid uptake is facilitating the transition of secretory cells into DATP-AT1 cells. This is an important question which authors should address. Another question that needs to answered is whether there is/are few specific essential amino acids that are responsible for the phenotype change observed here. Authors should consider demonstrating an actual increase in the uptake of essential amino acids (which may also help answer the second question above) as cells transition from secretory phenotype to DATP/AT1 phenotype. Moreover, the impact of mTORC1 inhibition on essential amino acid uptake in LATS1/2 dKO cells should be studied.
2) ATF4 expression is known to be pre-dominantly regulated at the translational level, owing to increased phosphorylation of eIF2-alpha. Did authors evaluate a possible change in ATF4 protein levels and p-eIF2-alpha levels (by western blotting)?
3) Authors should demonstrate the role of ATF4 in the upregulation of the amino acid transporter SLC7A5 and the consequent increased uptake of essential amino acids.
4) It remains unclear how the activation of YAP/TAZ signaling is increasing mTORC1 activity and consequently ATF4 transcription. Essential amino acids are known positive modulators of mTORC1 activity. Is it conceivable that ATF4-mediated upregulation of SLC7A5 precedes and facilitates essential amino acid uptake, which in turn enhances mTORC1 activity? It may be an important avenue to look into in order to develop a better understanding of the mechanistic underpinnings of the observations. 5) How do authors rule out that suppression of ATF4 in Raptor deleted cells is not simply due to compromised mRNA translation, owing to impaired mTORC1 activity?

Response to the Reviewers' comments
We are grateful for the reviewers' valuable comments, which helped us strengthen our manuscript. We have comprehensively addressed all concerns with additional experiments, which we believe in providing stronger and clearer support for our conclusions.

Referee #1:
This is an interesting manuscript. The phenotype of Lats1/2 dKO is carefully examined in the lung bronchial airway. In the Lats1/2 dKO, the columnar secretory cells lose their normal architecture and function, and are transformed into a flattened cell type that resembles alveolar AT1 cells. This fate conversion appears to involve TORC1 and ATF4. The human syndrome bronchiolitis obliterans appears similar to the Lats1/2 dKO phenotype.
Overall, the quantity and quality of data analysis are highly impressive, and the results will be of great interest to the Hippo signalling field, as well as to pulmonary science.

Response:
We thank the Reviewer for highlighting the novelty of our findings that advance the fields.
Minor comments 1. The human BO phenotype characterisation is important. Would the authors consider making this Figure 1 and mentioning it in the abstract, or even title of the manuscript? Response: Following the reviewer's suggestion, we have amended the abstract by highlighting the physiological relevance of our finding in BO phenotypes in our revised manuscript (In abstract, page; 2, line; 10-13).
2. It is interesting that the dKO organoids form multilayered epithelia, while in vivo the dKO forms a monolayer. Can the authors speculate as to why?
Response: As shown in Fig. 1B, D and Fig. 2G, H, we observed multi-layered epithelium at day 5 post Lats1/2 deletion. It is likely that YAP/TAZ activation at the early phase before converting into DATP cell states enhanced the proliferation of secretory cells. However, some of them, except differentiated DATPs and AT1 cells, detached off, leaving the epithelium monolayered. In contrast, secretory and differentiated cells in 3D organoids do not seem to detach significantly, possibly due to the different microenvironments such as the lack of mucociliary airway clearance in vitro.
We thank the Reviewer for all the constructive and helpful suggestions. In this manuscript, Jeon et al., used elegant genetic and pharmacological models to modulate Yap, mTOR and amino acid uptake mechanisms and claim that activation of Yap in airway secretory cells converts them to alveolar type-1 (AT1) cells via mTOR/amino acid import mechanisms. Specifically, the authors used genetic models to modulate Yap signaling in airway secretory cell and claim that activation of YAP in SCGB1A1+ airway secretory cells convert them into AT1-like cells in vivo and in ex vivo organotypic cultures. Additionally, the authors state that this plasticity involves a recently identified DATP state through which secretory cells transition to AT1 cells. The authors also claim that YAP-mediated essential amino acid influx controls secretory cell plasticity via mTOR signaling. Finally, the authors claim that similar mechanisms are at play in human lung diseases including pulmonary fibrosis and bronchiolitis obliterans. In summary, while the mechanisms described here are of potential importance to our understanding of airway cell plasticity, as I detailed below, the observations made here falls short of physiological relevance. Additionally, the data provided here does not support some of the claims (see below). Addressing the following comments would make this manuscript more meaningful and support the claims in this manuscript.
Major comments: 1. The authors show that activation of YAP/TAZ is sufficient to convert airway secretory cells into AT1 cells. However, plasticity observed here is somewhat "artificial" as the current data does not show whether the same mechanisms are involved in injury repair. Recent studies (Strunz et al., 2020) have indicated that Sox2-lineage labeled cells can generate AT2 and AT1 cells after bleomycin injury. Therefore, this reviewer questions the relevance of this finding to injury repair! Response: We fully agree with the Reviewer's critical comment related to the physiological relevance of our findings to injury repair. Interestingly, we observed a transient YAP/TAZ activation in lineage-labeled secretory cells of Scgb1a1-CreER TM/+ ;R26R tdTomato/+ mice during airway injury repair in response to naphthalene treatment. At day 5 post-injury, increased nuclear localization of YAP/TAZ was detected in lineage-labeled cells, which lost CC10 expression and acquired the expression of DATP marker CLDN4 (Revised Fig. EV5A, B). We also observed the expression of phospho-S6 and ATF4 in these cells, which was then declined with the regeneration of secretory cells (Revised Fig. EV5A, B). There was no AT1 cell differentiation on this occasion. These results suggest the potential role of YAP/TAZ activation induced by injury in modulating an emergence of DATPs with mTORC1/ATF4 activity during airway injury repair. Significantly, we observed Scgb1a1 + lineage-labeled AT1 cells in the airway epithelium post chronic airway injury by repetitive naphthalene treatment (Revised Fig.  EV5C, D). Sustained YAP/TAZ activation induced by chronic damage likely promotes fate conversion of secretory cells into AT1 cells, relevant to chronic lung diseases such as BO and IPF. Future study needs to be elucidated to understand the cellular function of DATPs emerging during acute injury repair in the airway. It would also be important to understand how the sustained YAP/TAZ activation induced by chronic injury results in the altered fate conversion of secretory cells into AT1 fate while there is no visible abnormal change in the acute injury model. We have now included these new data and discussed the physiological relevance of YAP/TAZ activation in the discussion section of our revised manuscript (page:13, line; 11- 21) 2. The secretory cell derived AT1 cells are located in airways, so, they may not have physiological relevance as these cells need to be connected to the vasculature for gas exchange. So, the question is whether there is micro-capillary associated with these AT1 cells? Additionally, prior studies have shown that airway secretory cells can migrate to alveolar regions after damage (ex: flu infection). So, how about activating Yap in secretory cells once they migrate to alveoli after injury (ex: flu infection) to test whether these cells can generate AT1? I suggest the author test this as this will reveal the physiological relevance of secretory cell derived AT1 cells.

Response:
We thank the reviewer for these critical comments. As suggested, we have carefully checked the vascular cells around AT1 cells derived from Lats1/2-deficient secretory cells. We found the microvascular structure expressing endothelial marker VECAM adjacent to lineagelabeled AT1 cells in the airways, suggesting that YAP/TAZ activation converted secretory cells into bona-fide AT1 cells retaining the potential for gas-exchange with capillary endothelial cells (Revised Fig. EV1E).
It would be interesting to test whether YAP-activated secretory cells can migrate and differentiate into AT1 cells in the alveoli post-injury. However, as we have shown, Lats1/2deficient secretory cells quickly transited into DATPs and AT1 cells at day 5 post tamoxifen treatment. They caused severe pulmonary fibrosis with the compromised epithelial integrity in the airways. Thus, we were unable to deliver a further injury to these animals due to the health concerns. Also, conducting the flu infection experiment requires special facilities and permission, but we did not have both facilities and permission. Instead, we have transplanted Scgb1a1 + lineage-labeled secretory cells isolated from Lats1/2 dKO lungs into bleomycin-injured WT lungs via intratracheal administration (Rev_Figure 1). Importantly, we observed lineage-labeled AT1 cells in the alveoli, indicating the differentiation ability of YAP/TAZ-activating secretory cells into AT1 cells. 3. The authors used the term DATPs. However, the plasticity observed here is due to genetic inactivation of Lats1/2 (and not due to injury/damage alone). It is more appropriate to use reprogrammed cells or Yap-induced transient state.

Response:
We used the terminology of DATP as the intermediate cell state emerging by YAP/TAZ activation from secretory cells before AT1 cell differentiation showed the transcriptional signatures significantly shared with DATPs emerging during injury repair. Thus, we want to use the term of DATPs in this paper.
4. The authors used tdTomato+ cells from Scgb1a1-creER/R26-tdtomato mice for scRNAsequencing ( Fig. 2 and EV3). However, there are numerous immune, fibroblast and mesothelial cells. This suggests that there are contaminating cells and that might obscure some of the findings (ex: organoid cultures). The authors need to provide additional data to support cell purity or repeat these experiments with pure cell populations.

Response:
As the Reviewer pointed out, cells acquired for scRNA-seq analysis showed some contamination of non-epithelial cells. So, we removed these clusters for analysis as there is no tdTomato expression in immune cells, fibroblasts, and mesothelial cells. Additionally, we have performed cytospin staining to verify the cellular composition in the population of tdTomato + cells isolated from Scgb1a1-CreER TM/+ ;R26R tdTomato/+ mice before further experiments including organoid cultures. We have confirmed that CC10 + secretory cells were mostly enriched in the population of tdTomato + cells (Rev_Figure 2).

Rev_Figure 2. Enrichment of secretory cells in tdTomato+ lineage-labeled cells.
Tomato + cells isolated from Scgb1a1-CreER TM/+ ;R26R tdTomato/+ lungs retain secretory cells. Representative IF images of cytospin staining from lineage-labeled cells isolated from Scgb1a1-CreER TM/+ ;R26R tdTomato/+ mice. CC10 (white), tdTom (red), and DAPI (blue). Scale bar, 100μm. Fig-2G' and Fig. EV2E: It appears that the authors used the same images (red and green channels) in both figures. Although this does not affect the conclusions in this manuscript, this might be flagged as "image duplication".

Response:
We apologize for the mis-organized figures. We have carefully checked and amended typos in our revised manuscript. 6. In Fig-3F, there are some tdTomato+ cells in EtOH treated condition (top right corner). Is that due to sporadic activation of cre recombinase or is this mouse line leaky?

Response:
As the Reviewer pointed out, we detected the low incidence of labeled secretory cells in corn oil-treated Scgb1a1-CreER TM/+ ; R26R fGFP/+ mouse lungs, consistent with an original paper generating this mouse model (Rawlins et al., 2009) (Rev_Figure 3). In order to minimize this effect, we used 4-6 weeks-old male mice, which display minimum leakiness.

Rev_Figure 3. Validation of the leakiness of reporter mouse model.
Representative IF images showing reporter expression after corn oil treatment in the indicated genotype: GFP (green), SPC (red), and DAPI (blue). Arrows point to report-positive cells. Scale bars, 50μm. 7. Generally, AT1 cells are localized to luminal side in most organoid cultures (Ex: Barkauskas et al., JCI 2013). However, in Fig-3D', AT1 markers are localized to basal side. Therefore, it is unclear whether the AT1-like cells that the authors observed in these models truly represent resident AT1 cells? Therefore, a comprehensive analysis of AT1-like cells in in vivo and ex vivo models and a direct comparison with resident AT1 cells is required to check the validity of the conclusions.
Response: As pointed out, AT1 cells retained in organoids derived from AT2 cells are localized luminal side (inner part) of organoids because AT2 cells can self-renew and also give rise to AT1 cells. However, in our study, YAP/TAZ activation in the secretory cells promotes the cellular conversion of secretory cells into AT1 cell fate via DATP cell states. Thus, AT1 cells were detected in the basal side of organoids derived from Lats1/2-deficient secretory cells. Furthermore, as the Reviewer suggested, we have confirmed the emergence of vasculature adjacent to lineage-labeled AT1 cells derived from Lats1/2-deficient secretory cells in the airways (Revised Fig. EV1E), and the differentiation potential of Lats1/2-deficient secretory cells into AT1 cells in the alveoli (Rev_Figure 1). These data support that AT1 cells derived from Lats1/2-deficient secretory cells are a bona-fide AT1 cell in our study with in vivo and ex vivo model. 8. In Fig. 5 and EV6, the authors claim that AT1-like cells are observed in airways of pulmonary fibrosis and bronchiolitis Obliterans lungs. However, the marker used here (AQP5) is not specific to AT1 cells. AQP5 is known to express in airway epithelial cells. Therefore, additional markers such as AGER and PDPN are necessary to support this claim. Additionally, the cells do not appear to show flat and thin morphology as expected for AT1 cells. So, this reviewer doubts the conclusion that they are AT1 cells.

Response:
To prove the presence of AT1 cells in the airways, we also used another AT1 cell marker, Caveolin-1 (CAV1), in addition to AQP5 (Revised Fig. EV4A). Regarding the morphology of AT1 cells in BO tissues, we further observed monolayered thin epithelium retaining mostly AT1 cells and replaced the images in Fig. 6 and Fig. EV4 of our revised manuscript.

9.
It is unclear what the authors are trying to show from the data in Fig. EV6A-C? There is no indication that neither AT1 nor DATP cells have originated from secretory cells. Again, the authors chose to label cluster #4 as DATPs. However, the original study (Haberman et al., Science Advances, 2020) have termed them basaloid cells. Altogether, the current data does not support whether de novo conversion of secretory cells into AT1 cells occurs in these diseases.
Response: Recent other studies, including ours (Choi et al., 2020;Kobayashi et al., 2020;Strunz et al., 2020), alongside Haberman et al., identified KRT8 high population (named as ADI, PATS, DATP) that show similar transcriptional signatures of Basaloid found in human IPF sample (Habermann et al., 2020). Thus, we named this population following those recent studies.
10. The authors claim that they identified KDR as a marker of secretory cells. There are other prior studies identified KDR as a marker and they (ex: Jiang et al., Developmental Cell, 2021) should be cited here.

Response:
We have cited this paper in our revised manuscript.
Minor: -In Fig-1B and D, it would be easier for the authors if the panels clearly show the mouse genotype.

Response:
We have amended those figures following the reviewer's suggestion in our revised manuscript.
We thank the Reviewer for all the constructive and helpful suggestions.

Summary:
The manuscript by Jeon et al., titled "Airway secretory cell fate conversion via YAP-mTORC1dependent essential amino acid metabolism" offers some mechanistic understanding of a differentiation program in airway secretory cells, implicating YAP/TAZ signaling and downstream engagement and requirement of mTORC1-ATF4-mediated essential amino acid uptake. Although overall the findings are interesting, the study is well-conceptualized and thoughtfully designed, with well-controlled experiments, additional experimental work is needed to substantiate the conclusions in regard to the involvement/role of mTORC1-mediated ATF4 upregulation and the consequent increase in the expression of SLC7A5 and essential amino acid uptake. Essentially, authors need to link (1) YAP/TAZ signaling with mTORC1 (at least experimentally implicate previously known link), (2) mTORC1 signaling with ATF4, and (3) essential amino acid uptake with the conversion of the airway secretory cells into AT1 cells.
Specific Comments: 1) It remains to be determined how essential amino acid uptake is facilitating the transition of secretory cells into DATP-AT1 cells. This is an important question which authors should address. Another question that needs to answered is whether there is/are few specific essential amino acids that are responsible for the phenotype change observed here. Authors should consider demonstrating an actual increase in the uptake of essential amino acids (which may also help answer the second question above) as cells transition from secretory phenotype to DATP/AT1 phenotype. Moreover, the impact of mTORC1 inhibition on essential amino acid uptake in LATS1/2 dKO cells should be studied.

Response:
We thank the Reviewer's valuable comments. Our study focused on defining how persistent YAP/TAZ activation caused altered cell fate conversion of airway secretory cells into squamous AT1 fate in the airways via mTORC1-ATF4 activity and its implication in human lung diseases such as BO and IPF. We further identified that EAA uptake via Slc7a5 is crucial for this fate transition via DATP cell state known to emerge during injury repair. We fully agree that determining how EAA uptake promotes this cellular change would be an important question in our future study. Our recent study showed that glycolytic metabolism is a key driver for cell fate transition into DATPs retaining the capacity to convert into AT1 cells (Choi et al., 2020). It has been also suggested that Slc7a5-dependent EAA uptake is required for glycolysis metabolism (Yoon et al., 2018;Yue et al., 2017). Thus, it is likely that metabolic realignment into glycolysis, mediated by EAA uptake, seems to be critical for fate decision of secretory cells. We will test this hypothesis in the future.
We have extensively tried to further narrow down the candidate EAA critical for fate conversion of secretory cells. Nine EAA (Histidine, Isoleucine, Leucine, Methionine, Threonine, Valine, Glutamine, Arginine, Cysteine) were tested in our study. As shown in Rev_Figure 4, the addition of branched chain amino acids (BCAA; Isoleucine, Leucine, Valine) in AA-limited BME media was not sufficient to promote fate conversion of secretory cells into DATPs. However, we observed the emergence of DATPs in organoids treated with conditional EAA (Arginine, Cysteine, Glutamine, Glycine, Proline, Tyrosine). Thus, it is likely that 4 amino acids (Cysteine, Arginine, Glutamine, and Glycine) seem to be essential components to drive the fate conversion of secretory cells into DATP/AT1 cells, which is regulated by YAP/TAZ activation. Further investigation would be interesting to study how these specific EAAs are regulated in this process in the future.
As suggested, we measured the actual increase of amino acid uptake in the cellular transition by isolating lineage-labeled cells from control, Lats1/2 dKO, and Lats1/2;Raptor tKO lungs at day 5 post tamoxifen treatment. We observed the increased uptake of amino acids in Lats1/2 dKO cells where secretory cells transited into DATPs/AT1 cells (Revised Fig. 4H). Importantly, deletion of Raptor significantly inhibited the amino acid uptake by YAP/TAZ activation, in accordance with inhibiting cellular transitions of secretory cells into DATPs/AT1 cells.
2) ATF4 expression is known to be predominantly regulated at the translational level, owing to increased phosphorylation of eIF2-alpha. Did authors evaluate a possible change in ATF4 protein levels and p-eIF2-alpha levels (by western blotting)?
Response: As there are a limited number of secretory cells in the lungs, western blotting is not available in our system. Instead, we have already detected increased protein levels of nuclear ATF4 expression both in organoids and lung tissues from mice and humans using immunostaining analysis in our manuscript. As suggested, we also examined the dynamic change of p-eIF2-alpha in protein levels using IF staining. As shown in Rev_Figure 5, there was no significant upregulation of p-eIF2-alpha expression in the airways during cell fate transition by YAP/TAZ activation.
3) Authors should demonstrate the role of ATF4 in the upregulation of the amino acid transporter SLC7A5 and the consequent increased uptake of essential amino acids.
Response: As suggested, we examined the effect of ATF4 in EAA uptake and fate decision of secretory cells and included these results in our revised manuscript (Revised Fig. 5). Consistent with Lats1/2 dKO lungs, constitutive activation of YAP signalling by overexpressing YAP 5SA mutant in the airway cell line enhanced the EAA uptake with increased expression of ATF4 and its target genes including Slc7a5 (Revised Fig. 5A-C). However, knockdown (KD) of ATF4 caused the defects in EAA uptake with reduced expression of Slc7a5 (Revised Fig. 5A, C). Furthermore, ATF4 KD in organoids derived from Lats1/2-deficient secretory cells impaired the fate conversion of secretory cells into DATPs and AT1 cells (Revised Fig. 5D-F). We also confirmed that Slc7a5 KD in Lats1/2-deficient secretory organoids inhibited their transition into DATPs and AT1 cells (Revised Fig. 5D-F). In contrast, sustained overexpression of ATF4 significantly promoted the fate conversion of secretory cells into DATPs and AT1 cells (Revised Fig. 5G-I). These results strongly support the functional role of ATF4 in upregulating SLC7A5 levels, allowing EAA uptake.
4) It remains unclear how the activation of YAP/TAZ signaling is increasing mTORC1 activity and consequently ATF4 transcription. Essential amino acids are known positive modulators of mTORC1 activity. Is it conceivable that ATF4-mediated upregulation of SLC7A5 precedes and facilitates essential amino acid uptake, which in turn enhances mTORC1 activity? It may be an important avenue to look into in order to develop a better understanding of the mechanistic underpinnings of the observations.

Response:
We agree with the Reviewer's critical view that it is important to determine the molecular mechanisms of EAA and YAP-mTOR-ATF4 circuit in cell fate conversion. YAP/TAZ has been shown to directly and/or indirectly influence mTORC1 activity (Tumaneng et al., 2012;Hansen et al., 2015;Hu et al., 2017). As pointed out, we cannot completely rule out the possibility that enhanced mTORC1 activity by YAP/TAZ activation results from upregulation of EAA uptake by ATF4-dependent Slc7a5 expression. However, as shown in Revised Fig. 4I, organoids derived from Lats1/2-deficient secretory cells showed that the expression level of phospho-S6 was not significantly affected by EAA depletion (4OHT+BME media) compared to control (4OHT+3D media). Thus, it is likely that the upregulation of mTORC1 activity in Lats1/2-deficient secretory cells seems to be directly affected by YAP/TAZ activation. However, we observed the augmented activity of mTORC1 activity by addition of EAA (4OHT+EAA+BME media) compared to organoids cultured in limited amino acid (4OHT+BME media), which indicating that there is a feedforward positive feedback loop by EAA on mTORC1 activity. We hope that the Reviewer understands the challenge to show direct evidence that ATF4-mediated amino acid uptake comes before mTORC1 activity due to this positive feedback loop.

5)
How do authors rule out that suppression of ATF4 in Raptor deleted cells is not simply due to compromised mRNA translation, owing to impaired mTORC1 activity?
Response: As the Reviewer pointed out, inhibition of mTORC1 by Raptor deletion in Lats1/2 dKO mice can impact global mRNA translational activity. Thus, we cannot rule out the possibility 100%. However, the deletion of Raptor enhanced the upregulation of CC10 expression in the secretory cells of Lats1/2 dKO mice alongside the reduction of ATF4 and other DATP-marker genes. Also, ATF4 KD in Lats1/2-deficient organoids impaired the fate conversion of secretory cells into DATPs and AT1 cells (Revised Fig. 5D-F), suggesting the critical role of ATF4 in this cell conversion. Thus, it is unlikely that the rescue of cell fate conversion by mTORC1 inhibition is simply due to the overall translational halt. We hope that this explanation satisfies the Reviewer.
Finally, we thank the Reviewer for all the constructive and helpful suggestions.

8th Feb 2022 1st Revision -Editorial Decision
Dear Dae-Sik, dear Joo-Hyeon, Thank you for submitting your revised manuscript (EMBOJ-2021-109365R) to The EMBO Journal. Your amended study was sent back to the three referees for re-evaluation, and we have received comments from all of them, which I enclose below. As you will see, the referees stated that the issues raised have been adequately addressed and they are broadly now in favour of publication, pending a minor revision.
Thus, we are pleased to inform you that your manuscript has been accepted in principle for publication in The EMBO Journal.
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Is there an estimate of variation within each group of data? 3-5 mice were used for each group to get significant values using student's t-test No samples were excluded Mice within the sample genotypes were randomized for chemical treatments

Manuscript Number: EMBOJ-2021-109365
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